This invention is directed to a microbial method for preparation of methyl anthranilate from naturally occurring methyl N-methyl anthranilate.
In recent years there has been a considerable consumer preference for more and better "all natural" flavors. To meet this demand, there is a need to develop new methods for securing natural aroma chemicals.
Concord grape (Vitis labrusca) flavors represent a type of product which has enjoyed widespread use in the food industry. Methyl anthranilate is known to be the major contributor to the typical aroma of this fruit, and it is a valuable ingredient in many other flavoring systems. Although methyl anthranilate is widely distributed in nature, its isolation is not generally economical due to the extremely low levels present. Therefore an alternate method for obtaining natural methyl anthranilate is required.
Although methyl anthranilate is not easily obtained from natural sources, methyl N-methylanthranilate (hereinafter dimethyl anthranilate) is readily recovered from petitgrain mandarin leaf oil (Citrus reticulata) in a high yield. Consequently it would be desirable to take advantage of this ready availability to produce methyl anthranilate by a N-demethylation method.
Microbially mediated N-demethylation reactions have been reported in species of Pseudomonas (Taylor, B. F., in Appl. Environ. Microbiol, 46:1286, 1983), Streptomyces (Yamano, et al, in Ann. Rep. Takeda Res. Lab, 21:88, 1963 also Bellet and van Thuong U.S. Pat. No. 3,520,778 in 1970), Arthrobacter (Kaczkowski, J., in Acta Soc. Botan.Polon., 28:677, 1959) Cunninghamella, Xylaria (Mitscher et al., in Experientia, 24:133, 1968), Claviceps (Voigt and Bornschein, in Pharmazie, 22:258, 1967) and Aspergillus (Funderburk et al., in Proc. Southern Weed Conf., 20:389, 1967). Various types of N-dealkylation reactions have been reported in the degradation of lignin by dry-rot fungi, including members of the genera Phanerochaete (Kuwahara et al, in J. Ferment. Technol., 62:237, 1984), Polyporus, Ganoderma. Fomitoosis (Reade and McQueen, in Can. J. Microbiol 29:457, 1983), Polystictus (Shimazono and Nord, in Arch. Biochem. Biophys., 87:140, 1960), Trametes and Fomes (Smolikova and Tichy, in Biologia. 5:211, 1976).
Despite these reports, however, the microbial demethylation of dimethyl anthranilate has not been reported because, for one reason, both the starting material and the product are highly toxic to most microorganisms. Moreover, the substrate stereochemistry causes difficulty because the microbiological demethylations are enzymatic and hence are highly selective. As a result, no reported microbiological reaction can be applied as a general method for demethylation of substituted amines.
Therefore, it is an object of the invention to develop a microbiologically mediated demethylation method which unexpectedly will produce reasonable yields of methyl anthranilate. Another object is the development of a microbiological method which is effective despite the toxicity of the starting material and product.